A variety of limitations impede the development of peptides for therapeutic use. For example, therapeutic peptides generally exhibit low stability in vivo and are often rapidly cleared, e.g., within several minutes to a few hours, via chemical or enzymatic degradation following their administration to a subject, i.e., before any therapeutic effect can be achieved. Consequently, low bioavailability can necessitate frequent administration of the peptide, often by injection, at fairly high doses to maintain activity. Such high doses can lead to undesired side effects. Furthermore, the delivery of therapeutic peptides can be restricted by the selective permeability of membrane barriers (e.g., intestinal and blood-brain barriers). To promote their delivery into cells, to increase their half-life, and/or to maintain their activities, polypeptides of interest, e.g., small therapeutic polypeptides, can be covalently coupled to carrier polypeptides, e.g., any of a variety of polypeptides that can have a high affinity for a specific ligand or group of ligands e.g., sugars, nucleosides, salts, amino acids, fatty acids, or other molecules. Carrier polypeptides typically facilitate the transport of such ligands, e.g., into subcellular compartments, in extracellular fluids (e.g., in the blood) or across cell membranes. Beneficially, carrier proteins can thus promote the delivery of covalently linked target polypeptides into cells, reduce their toxicity, and/or prolong their stability and/or activity following the administration of the carrier-target conjugate to a subject.
Current methods for chemically coupling small peptides to carrier polypeptides range from the use of non-specific reagents, e.g., glutaraldehyde or carbodiimide activated N-hydroxysuccinimide esters, to highly specific heterobifunctional crosslinkers that can circumvent the formation of carrier polypeptide-carrier polypeptide or target-polypeptide-target polypeptide conjugates. However, such reagents can only be used to modify a limited number of amino acid residues (e.g., amino acids comprising amine, keto, thiol, sulfhydryl, or carboxyl groups). Using such crosslinkers to conjugate a carrier polypeptide to a target polypeptide can perturb the conformation of the carrier polypeptide, the target polypeptide, or the resulting carrier-target polypeptide conjugate, thus decreasing the conjugate's stability, biological activity, pharmacokinetic activity, etc. Coupling reactions that make use of these crosslinking reagents can produce a heterogeneous population of carrier-polypeptide-target polypeptide conjugates, decreasing manufacturing efficiency and complicating quality control.
What are needed in the art are methods and compositions for the efficient, cost-effective, large-scale production of homogenous populations of carrier polypeptide-target polypeptide conjugates. The invention provides these and other needs, as will be apparent upon review of the following disclosure.